Skin, the outermost barrier of mammals, protects body from hazards in the environment and keeps fluids within the body. Understanding regulatory networks that dictate skin morphogenesis during embryonic development and self-renewal and differentiation of skin stem cells during adulthood will enhance our knowledge of normal skin development and of progression to various skin disorders including skin cancer. MicroRNAs are a family of non-coding small RNAs conserved from Caenorhabditis elegans to human. This newly discovered RNA species counts for 1-3% genes in mammalian genome. It is estimated that more than one third of protein-encoding mRNAs are regulated by microRNAs. Seveal microRNAs are also implicated in the initiation and progression of human cancer. However, the functions of microRNA in mammalian development remain largely unknown. In preliminary studies, I demonstrated that epidermis and hair follicle express discrete sets of microRNAs. Loss of all microRNA expression during embryonic skin development leads to abnormal evagination of hair follicles into the epidermis. These results, for the first time, provide direct evidence that differentially expressed microRNAs play essential roles skin development. In this proposal, I will pursue the initial studies and probe more deeply into this hitherto unexplored area of skin biology. Specifically, in the K99 phase, I will first utilize accurate isolation of specific skin lineages by fluorescen-actived cell sorting method and high-throughput microarray profiling of microRNA expression to idenfity differentially expressed microRNAs in specific epidermal and hair lineages. Then in the ROO phase, I will use in situ hybridization method to monitor the expression patterns of these microRNAs during skin [unreadable] development. This study will reveal differntial expression of microRNAs in high resolution and provide candidates for future functional studies. In the K99 phase, I will also elucidate the functions of a differentiation-related microRNA, miR-203, in epidermal differentiation with in vitro and in vivo approaches. This study will also serve as a paradigm for my future independent studies. In the ROO phase, I will establish an in vivo mouse model to identify the direct target of miR-203. This study will elucidate the molecular mechanisms of miR-203's functions and improve our understanding of microRNA target recognition. Collectively, these studies will also pave the way for me to transit towards an independent investigator. Together, my long-term interest is to elucidate molecular mechanisms governing mammalian development and self-renweal and differentiation of stem cells. Here, I employ skin as the model system to investigate functions of a family of small RNA molecules, microRNAs, in embryonic development. Such research will provide future basis for clinical use of skin stem cells in regenerative medicine and for developing methods for prevention and treatment of skin cancers. [unreadable] [unreadable] [unreadable] [unreadable]